Nearly 50 million Americans experience severe or chronic pain, costing about 60 billion dollars annually in lost productivity. About 22-26% of the general population of the United States state that the pain they experience is orofacial in nature, including toothaches, temporomandibular joint pain, and facial pain. Orofacial pain is generally ranked to be more severe than peripheral pain, partly due to dense innervation of orofacial structures. We have identified the enzyme cyclin dependent kinase 5 (Cdk5) as having a role in pain signaling as its enzymatic activity is upregulated downstream of inflammation. Even though Cdk5 activity is intriguingly induced following inflammation, we also needed to determine if this activity change, in turn, had an actual impact on pain processing. We therefore tested genetically engineered mice with either have increased or decreased Cdk5 activity for their reactions to painful stimuli. We initially employed traditional pain testing such as planter paw withdrawal to examine the responses of the mice. Mice genetically engineered to have increased Cdk5 activity display lower thresholds than their littermates in response to noxious heat and mechanical pain (hyperalgesia, which is more sensitivity to pain). In contrast, mice with decreased Cdk5 activity show reduced pain sensitivity (hypoalgesia). This behavioral testing, however, is just reflex based and examines only peripheral pain reactions, but we have now employed operant assays that are more amenable to testing orofacial responses while additionally using a conflict/reward paradigm. This allows the animals to make a choice whether to withdraw from an aversive stimulus or endure pain to achieve a reward. These behavioral tests additionally provide investigator-independent measurement of pain using automatically recorded behavior of the observed animals, so they incur less stress, and their behavior can be measured repeatedly in a non-biased fashion. Currently, our lab has three behavioral testing devices to measure mechanical, thermal, and chemical related pain in the orofacial region. Mechanical pain is measured using an Orofacial Stimulation Test (OST) that is designed so that the mice must stick their snout through a drinking window that can be equipped with abrasive wires to get access to the reward bottle containing 30% sucrose. Using a similar device to OST called OPAD (Orofacial Pain Assessment Device), we have also examined the effects of Cdk5 on thermal sensitivity, where a mouse must make contact with two thermodes to access the reward bottle. The thermodes can be set at noxious hot or cold temperatures. Lastly, chemical aversion can be tested using a device called the lickometer to measure the consumption of water spiked with offensive chemicals, including the pungent plant compounds capsaicin and mustard oil. With all of our new behavioral assays, we have basically shown that increased Cdk5 activity leads to orofacial hyperalgesia while decreased activity causes hypoalgesia. Part of the reason for Cdk5s ability to modulate pain perception is due to modification of pain transducing ion channels that respond to noxious stimuli, thereby causing depolarization of the neuron and subsequent neuronal firing. We identified transient receptor potential vanilloid receptor 1 (TRPV1), an ion channel that is activated by noxious heat and acidity, as a substrate of Cdk5. Enzymatic modification of TRPV1 by Cdk5 may partially account for the increased aversion to heat seen in mice with Cdk5 hyperactivity. Later, we identified two additional nociceptive ion channels as being substrates of Cdk5, the transient receptor potential ankyrin 1 (TRPA1), which acts as a chemosensor for the detection of environmental toxins, and the purinergic receptor P2X (P2X2a), which is involved in the detection of chemical signals released upon tissue injury. Cdk5 mediated modification of both of these ion channels may also play a role in accounting for the altered pain responses seen in our mice with either increased or decreased Cdk5 activity. Along with our behavioral testing of genetically modified mice, we have also engineered new mouse models that mimic aspects of known painful orofacial disorders. Inflammation is often the root cause in many instances of orofacial pain. We, therefore, wanted to better understand how inflammation is induced and to observe how it causes eventual destruction and functional loss in the affected tissues. The cytokine transforming necrosis factor-alpha (TNF-alpha) has long been known to cause recruitment and activation of immunoregulatory cells to the site of its secretion. Because of this established proinflammatory character, we decided to develop a strategy to genetically overexpress TNF- only within targeted tissues, thereby inducing a localized inflammation without the participation of an infectious agent that is typically associated with an immune response. We first developed a mouse model exhibiting painful pulpitis (inflammation in the dental pulp). We also overexpressed TNF- in pain sensing neurons, which mimicked some aspects of trigeminal neuralgia and caused increased aversion to orofacial heat in mice. Recently, we used this strategy to mimic sialadenitis, a painful condition characterized by salivary gland inflammation that is typically caused by either infection, sialolithiasis (salivary stones), or autoimmune disorders like Sjgrens syndrome. Since orofacial pain can often affect masticatory function, we tested these mice using another operant testing device known as the dolognawmeter, which measures the time needed for a mouse to gnaw through a plastic obstacle. With these mice, we were able to establish that overexpression of TNF-alpha just by itself was able to initiate an inflammatory cascade that lead to glandular destruction and difficulty in the ability to chew. With all of these mouse models, we were able to show that secretion of TNF-alpha can be major contributor to the pathology seen in many inflammatory diseases as just its expression alone was able to produce wide spread tissue damage in the targeted sites. In summary, our lab has been interested in understanding various aspects of orofacial pain, from developing new mouse models mimicking these painful conditions to identifying a key neuronal enzyme that can modulate pain sensitivity. We plan to examine if inhibiting Cdk5 activity may have a therapeutic effect and also plan to use additional models of orofacial pain to study the pain signaling pathway. Furthermore, we will continue our efforts to collaboratively study role of TGF-beta in disease processes affecting orofacial tissues.